A molecular spectrometer (sometimes referred to as a spectroscope) is an instrument wherein a solid, liquid, or gaseous specimen is illuminated, often with non-visible light, such as light in the infrared region of the spectrum. The light from the specimen is then captured and analyzed to reveal information about the characteristics of the specimen. As an example, a specimen may be illuminated with infrared light having known intensity across a range of wavelengths, and the light transmitted and/or reflected by the specimen can then be captured for comparison to the illuminating light. Review of the captured spectra (i.e., light intensity vs. wavelength data) can then illustrate the wavelengths at which the illuminating light was absorbed by the specimen, which in turn can yield information about the chemical bonds present in the specimen, and thus its composition and other characteristics. To illustrate, libraries of spectra obtained from reference specimens of known composition are available, and by matching measured spectra versus these reference spectra, one can then determine the composition of the specimens from which the measured spectra were obtained.
Two common types of spectrometers are dispersive spectrometers and Fourier Transform (FT) spectrometers. In a dispersive spectrometer, a range of input light wavelengths are supplied to a specimen, and the output light from the specimen is received by a monochromator—a device which breaks the output light into its component wavelengths—with one or more detectors then measuring light intensity at these output wavelengths to generate the output spectrum. In an FT spectrometer, an interferometer is used to supply an interferogram—a time-varying mixture of several input light wavelengths—to a specimen, and one or more detectors measure the (time-varying) output light from the specimen. The various wavelengths of the output light can then be “unscrambled” using mathematical techniques, such as the Fourier Transform, to obtain the intensity of the output light at its component wavelengths and thereby generate the output spectrum.
Spectroscopic microscopes then usefully incorporate the ability to make spectroscopic measurements into an optical microscope. A user may therefore use a spectroscopic microscope to view an image of a region of interest on a specimen (usually in magnified form), and also to obtain spectroscopic data from one or more locations on the region of interest. In some instances, the spectroscopic measurements are automatically collected by capturing spectroscopic data along 1-dimensional rows of areas on the region of interest (i.e., at areas spaced along a line on the region of interest), and then repeatedly capturing spectroscopic data from adjacent 1-dimensional rows. In other words, the linear array of spectroscopically-sampled areas is stepped sideways to ultimately capture spectroscopic data over a 2-dimensional array of areas over the region of interest. As a result, the user can view an image of the region of interest, and can also review the spectra (and thus the composition) of the specimen at locations arrayed over the region of interest. A disadvantage of this approach is that each spectrum captured from an area will reflect the presence of whatever substances are present in the area (or at least the presence of those substances that are responsive to the incident light): for instance, if an area includes a particle of particular interest to the user, the captured spectrum from the area will reflect not just the spectrum of the particle, but will also tend to include contributions from the substances surrounding the particle within the area. The results of such measurements can still be useful—a user might still obtain information regarding the particle—but the results must be carefully interpreted.
As an alternative, users can manually collect spectroscopic readings from specimens. As an example, a user interested in obtaining data on the aforementioned particle might limit the field of view of the spectroscope's detector to only the area of the particle, as by masking the specimen such that only the particle is visible to the detector through an aperture. The spectrum captured from the aperture will then reflect only the contributions of the substances “seen” by the detector through the aperture. Masks with variable apertures are commonly used in spectroscopic microscopes, with the mask having two stacked pairs of plates, wherein the plates within each pair are coplanarly and adjacently situated with a space between their adjacent edges, and wherein the edges of the pairs are perpendicularly oriented with respect to each other. The plates within the pairs are then movable, usually in synchronized fashion, so that the space between the plate edges within each pair can be controlled. As a result, moving the plates within the pairs together, or moving them apart, generates an aperture with a square area of variable size. The disadvantage of the manual approach is that it is time-consuming and tedious—a user must move the mask to each particle of interest, and size the aperture so that only the desired area is imaged to the detector—and here too results must be carefully interpreted. For example, many beginning users of spectroscopic microscopes fail to appreciate that if a particle is in the nature of an occlusion within the specimen (e.g., as viewed, it is covered by an invisible layer of another substance), the spectrum captured from the particle will reflect not only the substance(s) present within the particle, but also those in the covering layer. Generating the correct settings for the microscope can also be a challenge, since many novice users fail to appreciate that the image of the specimen seen through the microscope's eyepiece(s) or camera may not correspond to the specimen as seen by the spectroscopic detector; thus, simply focusing on a particle or the like within a specimen may not limit the detector's field of view (and thus the spectroscopic readings) to the particle. Other subtleties also tend to hinder novice users, such as the issue that a smaller aperture setting (smaller detector field of view) tends to decrease the signal-to-noise ratio of the resulting spectral readings simply because the detector captures less light from a smaller field of view. Thus, smaller aperture settings usually require a longer exposure time (i.e., a higher detector data collection time), and/or the use of multiple exposures, which can then be combined (as by averaging them) to decrease the effects of noise.
All of the foregoing issues tend to pose obstacles to effective use of spectroscopic microscopes, particularly to new users. It would therefore be useful to have spectroscopic microscopes, and methods of operating spectroscopic microscopes, which allow for easier and more accurate collection of spectral data from particular areas within a region of interest on a specimen.